aprepitant and Hepatoblastoma

The substance P (SP)/NK-1 receptor (NK1R) complex represents an intriguing anticancer target for a variety of tumors, including hepatoblastoma (HB). Therefore, NK1R antagonists, such as the clinical drug aprepitant, recently have been proposed as potent anticancer agents. However, very little is known regarding the molecular basis of NK1R inhibition in cancer. Using reverse phase protein array, Western blot, Super TOP/FOP, confocal microscopy, and sphere formation ability (SFA) assays, we identified the AKT and Wnt signaling pathways as the key targets of aprepitant in three human HB cell lines (HepT1, HepG2, and HuH6). Following NK1R blockage, we observed decreased phosphorylation of p70S6K and 4E-BP1/2 and inhibition of the canonical Wnt pathway with subsequent decrease of HB cell growth. This effect was dependent of high baseline Wnt activity either by mutational status of β-catenin or extrinsic Wnt activation. Wnt inhibition seemed to be strengthened by disruption of the FOXM1-β-catenin complex. Furthermore, treatment of HB cells with aprepitant led to reduced expression of (liver) stemness markers (AFP, CD13, SOX2, NANOG, and OCT4) and SFA when grown under cancer stem cell conditions. Taken together, we show for the first time that targeting the SP/NK1R signaling cascade inhibits canonical Wnt signaling in HB cells. These findings reveal important insight into the molecular mechanisms of the SP/NK1R complex as a critical component in a model of pediatric liver cancer and may support the development of novel therapeutic interventions for HB and other Wnt-activated cancers.

Topics: Aprepitant; beta Catenin; Cell Line, Tumor; Cell Proliferation; Child; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Hepatoblastoma; Humans; Liver Neoplasms; Morpholines; Oncogene Protein v-akt; Receptors, Neurokinin-1; Wnt Signaling Pathway

Multidrug resistance presents a major problem in hepatoblastoma (HB), and new anti-tumor strategies are desperately needed. The substance P (SP)/neurokinin-1 receptor (NK1R) complex has been discovered to be pivotal in the development of a variety of human cancers, and NK1R antagonists, such as the clinical drug aprepitant, are promising future anticancer agents. Yet, the role of the SP/NK1R complex as a potential anticancer target in HB is unknown.. Human HB cell lines HepT1, HepG2, and HuH6, human tumor samples from 17 children with HB as well as mice xenografted with human HB cell line HuH6 were analyzed regarding the SP/NK1R complex as a potential new anti-tumor target in HB.. Therapeutic targeting with the NK1R antagonists aprepitant, L-733,060, and L-732,138 led to growth inhibition and apoptosis in HepT1, HepG2, and HuH6 cells in a dose-dependent manner. Intriguingly, HB cells predominantly expressed the truncated splice variant of NK1R. Human fibroblasts showed only dismal NK1R expression and were significantly more resistant. Stimulation of HB cells with SP, NK1R's natural ligand, caused increased growth rates and abrogated the anti-proliferative effect of NK1R antagonists. Expression analysis of 17 human HB samples confirmed the clinical relevance of NK1R. Most importantly, oral treatment of a HuH6 xenograft mouse model with 80mg/kg/day aprepitant for 24days resulted in a striking reduction of tumor growth, as evidenced by reduced tumor volume and weight, lowered tumor-specific alpha-fetoprotein (AFP) serum levels, and decreased number of Ki-67 positive cells. Furthermore, aprepitant treatment inhibited in vivo angiogenesis.. For the first time, we describe the NK1R in its truncated splice variant as a potent target in human HB and an inhibitory effect in vivo and in vitro by NK1R antagonists. Therefore, NK1R antagonists should be considered promising new candidates for innovative therapeutic strategies against HB.

Topics: Animals; Apoptosis; Aprepitant; Cell Line, Tumor; Cell Proliferation; Child; Cytostatic Agents; Drug Synergism; Female; Gene Expression; Hep G2 Cells; Hepatoblastoma; Humans; Liver Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Morpholines; Neovascularization, Pathologic; Neurokinin-1 Receptor Antagonists; Protein Isoforms; Receptors, Neurokinin-1; Substance P; Xenograft Model Antitumor Assays